American Journal of Plant Sciences, 2015, 6, 1697-1712 Published Online July 2015 in SciRes. http://www.scirp.org/journal/ajps http://dx.doi.org/10.4236/ajps.2015.611169
Patterns of Molecular Diversity in Current and Previously Developed Hybrid Parents of Pearl Millet [Pennisetum glaucum (L.) R. Br.] Shashi Kumar Gupta1, Thirunavukkarasu Nepolean2, S. Mukesh Sankar2, Abhishek Rathore1, Roma Rani Das1, Kedar Nath Rai1, Charles Thomas Hash3 1
International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Patancheru, India Indian Agricultural Research Institute (IARI), New Delhi, India 3 International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Niamey, Niger Email:
[email protected] 2
Received 17 April 2015; accepted 12 July 2015; published 15 July 2015 Copyright © 2015 by authors and Scientific Research Publishing Inc. This work is licensed under the Creative Commons Attribution International License (CC BY). http://creativecommons.org/licenses/by/4.0/
Abstract ICRISAT’s pearl millet (Pennisetum glaucum (L.) R. Br.) breeding program at Patancheru, India, has developed genetically diverse hybrid parents since 1980s. The present study investigated genetic diversity pattern between two groups of parents in this program, bred till 2004 and developed during 2004-2010. Combined analysis of 379 hybrid parents (current 166 parents and 213 previously developed hybrid parents) carried out using a set of highly polymorphic 28 SSRs detected 12.7 alleles per locus. An average of 8.5 and 8.7 SSR alleles per locus were found in previously developed and current parents, respectively, indicating marginal improvement in the levels of genetic diversity of hybrid parents in this program. Distance matrix differentiated these current and previously developed hybrid parents into 2 separate clusters, indicating infusion of new genetic variability over time as reflected by development of more genotype-specific alleles. Also, the seed and restorer parents were found clearly separated from each other in both the sets with few crossovers, indicating existence of two diverse and broad-based pools in hybrid parents of pearl millet. Restorer parents (R-lines) were found more diverse than seed parents (B-lines), as higher average gene diversity was detected among R-lines (0.70) than B-lines (0.56), though variation between Band R-lines was found reduced in newly developed lines to 9.22% from 16.98% in previously developed lines. Results suggested that newly developed lines were as much divergent when compared with previously developed lines, indicating that current ICRISAT pearl millet breeding program was moving towards development of diverse new hybrid parental lines. The study suggested use of trait-specific donors in B- and R-lines separately to maintain sufficient genetic distance between seed and restorer breeding lines. It was pointed out to cross parents having higher genetic distance within the seed (B-lines) and restorer (R-lines) breeding programs to derive diverse and productive hybrid parental lines in future. How to cite this paper: Gupta, S.K., Nepolean, T., Sankar, S.M., Rathore, A., Das, R.R., Rai, K.N. and Hash, C.T. (2015) Patterns of Molecular Diversity in Current and Previously Developed Hybrid Parents of Pearl Millet [Pennisetum glaucum (L.) R. Br.]. American Journal of Plant Sciences, 6, 1697-1712. http://dx.doi.org/10.4236/ajps.2015.611169
S. K. Gupta et al.
Keywords Pearl Millet, Simple Sequence Repeat, Hybrid Parents, Heterotic Pool, Genetic Diversity
1. Introduction Pearl millet (Pennisetum glaucum (L.) R. Br.) is cultivated on about 30 million ha in more than 30 countries with the majority of this area in Asia (>10 million ha), Africa (about 18 million ha), and >2 million ha in the Americas. It is important as a high temperature and drought tolerant grain crop in Africa and Asia, particularly on low-fertility soils with limited water and nutrient holding capacity and is equally important as a hot season forage crop in semi-arid and arid parts of the world [1]. In Americas, it is used primarily as temporary summer pasture or as mulch in no-till soybean production systems [2]. India is the largest producer of this crop with 9.6 million ha area and 10 million tons of grain production [3]. The productivity of pearl millet in India rose from 305 kg∙ha−1 during pre-hybrid era (1951-1955) to 1040 kg∙ha−1 during present hybrid dominated era (2008-2013), and registered 227% improvement [4]. Hybrid technology contributed significantly to this increase in grain yield performance in India, and currently about 70% of the pearl millet area in India is sown to hybrids. Based on the expected significant contribution of hybrids to pearl millet productivity enhancement in India, in the 1980s ICRISAT-Patancheru re-aligned its research strategy to better complement those of the Indian national program and private seed industry, and so focused on genetic diversification of hybrid parents. A recently conducted study found that private seed companies developed 103 hybrids in India during 2000-2010, and 62 (60%) were directly or indirectly based on ICRISAT bred hybrid parents [5]. Genetic diversification of hybrid parents has been the key to developing the diverse range of hybrids required to adapt under different agro-ecologies in India. Hence, ICRISAT-Patancheru’s pearl millet hybrid breeding program utilizes new genetic stocks regularly in the crossing program to widen the genetic diversity of hybrid parents. Hence, in the late 1990s and early 2000 years, there was an increase in the use of already designated hybrid parents and newly developed composites in ICRISAT pearl millet hybrid parent’s development crossing program to derive new wave of genetically diverse inbreds. In this breeding process, it is important to monitor the levels of genetic diversity among these newly developed promising hybrid parents, and SSRs being environment independent multi-allelic genomic tools offer considerable advantages for this. Hence, the present study was undertaken to assess the genetic diversity in 166 newly developed hybrid parents (yet to be designated 88 B-lines and 78 R-lines, drawn from ICRISAT-Patancheru’s hybrid breeding program in 2010) using a subset of highly polymorphic 28 SSR loci as suggested by previous studies [6]-[11] and results were compared with those for 213 previously developed hybrid parents (98 B-lines and 113 R-lines, developed during 1981 to 2004) to provide information on trends for genetic diversity in this breeding program over time.
2. Materials and Methods 2.1. Plant Material Two sets of hybrid parents, the first set (set-I of 213 hybrid parental lines comprising of 98 seed parents and 115 restorer parents, designated between 1984 to 2004 at ICRISAT-Patancheru), and a second set (set-II) of 166 current hybrid parents comprising of 88 seed parents (numbered from B-1 to B-88) and 78 restorer parents (numbered from R-89 to R-166) bred by the hybrid parent breeding program of ICRISAT-Patancheru and available in 2010, were used in the present study. The set-I parents were characterized for morphological traits [12] [13] and using molecular markers [14]. Tift 23D2B1, used as a reference genotype, was bred at the Coastal Plain Experiment Station, Tifton, GA, by introducing the d2 dwarfing gene into the genetic background of an elite line Tift 23B1, which is a maintainer of the A1 cytoplasmic-nuclear male sterility (CMS) system [15].
2.2. DNA Extraction The B- and R-lines along with Tift 23D2B1 were grown in small plastic pots in a greenhouse. Approximately 30 mg of leaf tissue from 20 - 25 seedlings (14 d old) were collected and bulked after removing the leaf tips and midribs. The harvested leaf samples were immediately collected in 96-well plate that consisted of 95 lines and
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one control (Tift 23D2B1). Genomic DNA was extracted following the standard protocol [16]. Quantification of DNA, quality check and normalization up to 5 ng∙μl−1 were done on agarose gel (1.2%, containing ethidium bromide).
2.3. Molecular Markers A set of 23 genomic- and five expressed sequence tag-simple sequence repeat (SSR) primer pairs were selected (based on the high level of PIC detected in earlier studies), to genotype the B- and R-lines of both the sets (set-I and set-II) using an ABI 3700 DNA fragment analyzer (Applied Biosystems). Twenty-five of these 28 SSRs mapped on pearl millet genome: 4 to 7 SSR loci on linkage groups (LGs) 1, 2, 3, and 7; and 2 SSR loci each on LGs 5 and 6 [6] [17].
2.4. Polymerase Chain Reaction (PCR) and Genotyping Polymerase chain reactions (PCRs) were performed in 5 μl volumes in 384-well PCR plates (AB Gene Rochester). Each PCR reaction mixture contained 5 ng of genomic DNA, 2 pmol/μl of each primer, 25 mM MgCl2, 2 mM of each deoxyribonucleotide triphosphate, 10× reaction buffer, and 0.2 U Amplitaq Gold Polymerase (Applied Bio-systems, India). After one denaturing step of 15 min at 94˚C, a touchdown amplification program was performed on GeneAmp 9700 thermal cycler (Applied Biosystems). This profile consisted of a denaturing step of 25 sec at 94˚C and an extension step of 30 sec at 72˚C. The initial annealing step was 20 sec at 64˚C for one cycle and subsequently the temperature was reduced by 1˚C for every cycle until a final temperature of 55˚C was reached. The annealing temperature of 55˚C was maintained for the last 35 cycles of the amplification, followed by the final extension of 72˚C for 7 min. PCR products were pooled post-PCR, which contained 1 μl of each of dye-labeled (FAM, VIC, NED and PET) product, 7 μl of formamide, 0.3 μl of the LIZ-labeled [500 (−250)] size standard, and 4.2 μl of distilled water. The DNA fragments were size-separated on ABI 3700 automatic DNA sequencer (Perkin-Elmer/Applied Biosystems). GeneScan 3.1 [18] was used to size peak patterns by using the internal LIZ-labeled (500 [−250]) size standard and Genotyper 3.1 [19] was used for allele calling. Final bins for individual marker were assigned with the help of marker repeat length using AlleloBin 2.0 program [20] and the binned data was used for further data analysis.
2.5. Data Analysis Genotypic data for the 28 common SSR markers was analyzed collectively for all the 379 lines (213 lines of set-I and 166 lines of set-II), and also separately for set-I and set-II lines. PIC; allelic richness as determined by total number of alleles, alleles per locus, and occurrence of common, most frequent, rare, and unique alleles; gene diversity; and heterozygosity were estimated using the PowerMarker V3.0 software [21]. Unique alleles are those that are present in one line but absent in other lines. Rare alleles are defined as those whose frequency is ≤1% in the investigated materials. Common alleles are defined as those whose frequency is 1% to 20% while the most frequent alleles are those whose frequency is >20%. This classification of alleles was done following method used by other workers [22] [23]. AMOVA was performed [24] for both the sets individually and also pooled analysis to estimate the variance components among and within B- and R-line groups. The F value—the fixation index (or Wright’s F statistic) (Fst)—from the AMOVA analysis provided a measure of genetic differentiation of the sub-groups. A simple matching allele frequency-based distance matrix was used in the DARwin-5.0 program [25] to construct a tree diagram to examine the genetic structure and diversity among B- and R-lines. The grouping of B- and R-lines into clusters and sub-clusters was done at 5% dissimilarity level.
3. Results In the combined set of 379 hybrid parents, comprising of both previously designated (hereafter, referred to as set-I) as well as current (hereafter, referred as set-II) pearl millet hybrid parents, 355 alleles were detected by 28 SSR loci, with an average of 12.68 alleles per locus. The number of alleles per locus varied from 4 (Xpsmp2202, Xpsmp2246 & Xipes3048) to 40 (Xpsmp2218), with ten to twenty alleles at 15 of the 28 SSR loci (Table 1). Marker Xpsmp2218 had up to 40 alleles while Xpsmp2068, Xpsmp2079 and Xpsmp2218 amplified 23 to 40 alleles per locus. The allele size range across the loci and lines varied from 4 bp (Xpsmp2201) to 121 bp
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(Xpsmp2079). Polymorphic information content, (PIC) ranged from 0.26 (Xpsmp2222) to 0.93 (Xpsmp2218) with an average of 0.67. Of the 28 SSRs used, 25 were moderately polymorphic (PIC > 0.44), of which 12 were highly polymorphic (PIC > 0.70) with PIC values ranging from 0.70 to 0.93. Furthermore, the average PIC values for B- and R-lines were 0.56 and 0.70, respectively. Gene diversity, defined as the probability that two randomly chosen alleles from the population are different, varied from 0.28 (Xpsmp2222) to 0.93 (Xpsmp2218) with an average of 0.70. Restorer lines (R-lines) had higher average gene diversity (0.73) than B-lines (0.60). The level of heterozygosity in SSRs across B- and R-lines ranged from 0.01 to 0.07 (excluding for Xpsmp2077 which had high value of 0.12) and averaging 0.04, with >0.05 in eight SSRs and >0.10 in one SSR. The R-lines had higher average heterozygosity (0.05) than B-lines (0.03). Moreover, ICMB 96666, B-23, B-24, B-57, B-65, B-6, B-45 and B-46 among B-lines; and ICMP 451, IPC 337, IPC 492, IPC 962, IPC 997, IPC 1018, IPC 1027, IPC 1078, IPC 1307, IPC 1503, IPC 1617, R-118, R-125, R-128, R-131, R-144, R-148, R-155, R-159, R-160, and R-166 among R-lines were genetically more variable than other lines, with heterozygosity at four to eight SSR loci detected each in the B-lines and in the R-lines (data not presented). Allele size for the internal control Table 1. Allelic composition, polymorphic information content (PIC), gene diversity, and observed heterozygosity of the 28 simple sequence repeat (SSR) loci in combined set of pearl millet (186 B-lines and 193 R-lines). Rare Common Most alleles alleles frequent (≤1%) (1 to ≤20%) alleles (>20%)
SSR loci
Allele richness
Allele size range (bp)‡
Xpsmp2045
10
21 (195 - 216)‡
7
1
Xpsmp2068
23
42 (99 - 141)
10
12
Xpsmp2077
9
40 (138 - 178)
3
Xpsmp2079
33
121 (138 - 259)
Xpsmp2089
20
Xpsmp2090 Xpsmp2201
PIC†
Gene diversity†
Heterozygosity†
2
0.51 (0.37, 0.54)
0.59 (0.41, 0.60)
0.02 (0.01, 0.03)
1
0.87 (0.76, 0.89)
0.88 (0.78, 0.90)
0.03 (0.04, 0.02)
4
2
0.51 (0.47, 0.43)
0.58 (0.56, 0.46)
0.12 (0.08, 0.15)
11
22
0
0.91 (0.85, 0.94)
0.92 (0.86, 0.94)
0.04 (0.02, 0.06)
30 (103 - 133)
4
15
1
0.88 (0.78, 0.91)
0.89 (0.81, 0.92)
0.05 (0.07, 0.04)
10
20 (171 - 191)
2
6
2
0.74 (0.70, 0.77)
0.78 (0.74, 0.79)
0.008 (0.01, 0.01)
10
4 (332 - 336)
2
6
2
0.66 (0.50, 0.75)
0.70 (0.59, 0.78)
0.02 (0.01, 0.03)
Xpsmp2202
4
18 (145 - 163)
0
1
3
0.59 (0.59, 0.59)
0.66 (0.67, 0.65)
0.03 (0.02, 0.04)
Xpsmp2203
19
37 (335 - 372)
4
13
2
0.86 (0.66, 0.91)
0.87 (0.70, 0.92)
0.03 (0.01, 0.06)
Xpsmp2204
11
94 (173 - 267)
3
7
1
0.58 (0.26, 0.77)
0.60 (0.30, 0.80)
0.02 (0.01, 0.03)
Xpsmp2209
12
23 (337 - 360)
4
5
3
0.82 (0.79, 0.79)
0.84 (0.81, 0.81)
0.07 (0.04, 0.10)
Xpsmp2218
40
59 (217 - 276)
19
21
0
0.93 (0.87, 0.94)
0.93 (0.88, 0.94)
0.03 (0.01, 0.05)
Xpsmp2220
12
24 (108 - 132)
4
7
1
0.70 (0.41, 0.83)
0.72 (0.43, 0.85)
0.04 (0.01, 0.07)
Xpsmp2222
5
8 (152 - 160)
0
4
1
0.26 (0.23, 0.30)
0.28 (0.24, 0.31)
0.05 (0.08, 0.02)
Xpsmp2227
5
15 (194 - 209)
2
2
1
0.28 (0.25, 0.30)
0.31 (0.28, 0.34)
0.02 (0.01, 0.03) 0.02 (0.02, 0.02)
Xpsmp2232
16
24 (226 - 250)
7
7
2
0.77 (0.65, 0.78)
0.80 (0.69, 0.80)
Xpsmp2237
10
56 (202 - 258)
5
4
1
0.56 (0.21, 0.73)
0.60 (0.22, 0.77)
0.04 (0.03, 0.04)
Xpsmp2246
4
6 (258 - 264)
0
2
2
0.68 (0.56, 0.70)
0.73 (0.63, 0.75)
0.03 (0.03, 0.02)
Xpsmp2248
6
10 (162 - 172)
0
4
2
0.70 (0.73, 0.64)
0.74 (0.77, 0.69)
0.02 (0.03, 0.02)
Xpsmp2249
6
31 (129 - 160)
2
3
1
0.39 (0.26, 0.49)
0.43 (0.28, 0.55)
0.02 (0.01, 0.03)
Xpsmp2273
19
62 (158 - 220)
9
9
1
0.77 (0.71, 0.79)
0.78 (0.73, 0.81)
0.04 (0.02, 0.05)
Xipes3002
10
14 (197 - 211)
4
5
1
0.65 (0.63, 0.65)
0.68 (0.67, 0.69)
0.05 (0.02, 0.07)
Xipes3032
6
15 (180 - 195)
2
2
2
0.63 (0.57, 0.57)
0.68 (0.63, 0.64)
0.07 (0.05, 0.08)
Xipes3048
4
6 (244 - 250)
0
2
2
0.49 (0.47, 0.50)
0.57 (0.56, 0.57)
0.008 (0.017, 0)
Xipes3080
7
15 (211 - 226)
3
2
2
0.63 (0.57, 0.58)
0.68 (0.62, 0.64)
0.05 (0.03, 0.04)
Xipes3088
11
24 (144 - 168)
2
8
1
0.83 (0.77, 0.80)
0.85 (0.80, 0.82)
0.03 (0.05, 0.01)
CTM10
17
32 (167 - 199)
3
13
1
0.78 (0.46, 0.91)
0.80 (0.50, 0.91)
0.05 (0.01, 0.10)
CTM12
16
25 (318 - 343)
5
9
2
0.75 (0.64, 0.77)
0.78 (0.67, 0.80)
0.03 (0.04, 0.02)
0.67 (0.56, 0.70)
0.70 (0.60, 0.73)
0.04 (0.03, 0.05)
Total
355
117
196
42
Mean
12.68
4.18
7
1.5
†
‡
PIC, gene diversity, and heterozygosity for the B- and R-lines are given in parentheses, respectively; Numbers in parentheses indicate range.
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(Tift 23D2B1) were uniform and reproducible for each of the markers indicating the accuracy of the protocol and reproducibility of allelic data for a given primer across assays. Of the 355 alleles detected in B- and R-lines, 117 were rare (frequency ≤ 1%), 196 common (frequency 1% to 20%), and 42 most frequent (frequency > 20%) alleles (Table 1), with more alleles observed in R-lines (329 alleles in 193 lines averaging 1.70 alleles per line) than the B-lines (237 alleles in 186 lines averaging 1.27 alleles per line). “Most frequent” alleles were detected at all the SSR loci (except in SSR loci, Xpsmp2079 and Xpsmp2218), ranging from one to three and averaging 1.5 alleles per locus. The rare alleles ranged from 0 (Xpsmp2202, Xpsmp2222, Xpsmp2246, Xpsmp2248 and Xipes3048) to 19 (Xpsmp2218), averaging 4.18, while the common alleles ranged from 1 (Xpsmp2045 and Xpsmp2202) to 22 (Xpsmp2079), averaging 7.0. Twenty-one SSR loci detected all three (rare, common, and most frequent) classes of alleles (Table 1). Genotype-specific alleles were detected in 15 B-and 37 R-lines (Table 2). The combined Analysis of Molecular Variance (AMOVA) for all the lines showed significant differences between set-I and set-II, which accounted for 19.20% of the total genetic variation (Table 3). Also, significant Table 2. Genotype-specific alleles present in 15 seed parents (B-lines) and 37 restorer parents (R-lines) of pearl millet (from both the sets). Accession identity†
SSR allele‡
ICMB 91444
Xpsmp2209353
B-13
Xpsmp2079138
ICMB 91666
Xpsmp2079259
B-33
Xpsmp2218234
ICMB 92444
Xpsmp2218217
B-37
Xpsmp2045216
ICMB 96222
Xpsmp2068137
B-44
Xpsmp2218276
ICMB 98333
Xpsmp2232226
B-59
Xipes3002208
B-49
Xpsmp2273188
Accession identity
SSR allele
Seed parents (B-lines)
B-61
Xpsmp2227197
B-63
Xpsmp2237234
B-64
Xpsmp2068102
B-66
Xpsmp2218268
Restorer parents (R-lines) IPC 244
Xpsmp2204191
R-94
Xpsmp2273186
IPC 338
CTM10169
R-96
Xpsmp2237202 and Xpsmp2273194 Xpsmp2045203
IPC 367
Xpsmp2203343
R-98
IPC 487
Xpsmp2204175
R-100
Xpsmp2079224
IPC 632
Xpsmp2220132
R-103
Xpsmp2201345
IPC 687
Xpsmp2232242
R-121
Xpsmp2218238
IPC 689
CTM12335
R-122
Xpsmp2089111
IPC 774
Xpsmp2232250
R-131
Xpsmp2218270
IPC 821
Xpsmp2203361
R-136
Xpsmp2045209
IPC 962
Xpsmp2273174
R-138
Xpsmp2218250
IPC 1027
Xpsmp2218247
R-140
Xpsmp2209342
IPC 1047
Xpsmp2077142
R-141
Xpsmp2079215
IPC 1114
Xpsmp2220114
R-146
Xpsmp2089132
IPC 1178
Xpsmp2249150
R-149
Xipes3088168
IPC 1254
Xpsmp2273176
R-158
CTM12324
IPC 1307
Xpsmp2079235
IPC 1329
Xpsmp2273180
IPC 1351
Xpsmp2068129
IPC 1445
Xpsmp2079219
IPC 1536
CTM10199
†
ICMB, ICRISAT millet B-line; IPC, ICRISAT pollinator (restorer) collection; ‡SSR, simple sequence repeat.
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differences were observed between B- and R-line groups within each set, which accounted for 11.55% of the total genetic variation. The within-group variation, both in the B-lines and R-lines, was much larger and accounted for 69.24% of the total observed genetic variation. Set-wise AMOVA showed higher variation between B- and R-line groups in set-I (16.98%) than in set-II (9.92%), as detected by 28 SSR loci. The fixation index (Fst) between B- and R-lines for the individual markers varied from 0.138 (Xpsmp2222) to 0.456 (Xpsmp2218) and was significant for all 28 SSR primer pairs (information not presented). Some of the markers such as Xipes3088, Xpsmp2209, Xpsmp2203, Xpsmp2068, Xpsmp2089, Xpsmp2079 and Xpsmp2218 had Fst values greater than 0.40, contributed most to the variation between B- and R-line groups. Mean for number of alleles per locus, gene diversity, heterozygosity and PIC for set-I using the set of 28 SSRs was 8.50, 0.63 (0.50 for B-lines and 0.63 for R-lines), 0.03 (0.02 for B-lines and 0.04 for R-lines), and 0.59 (0.46 for B-lines and 0.60 for R-lines) respectively (Table 4). In set-II of current pearl millet hybrid parents, 28 SSR loci detected a total of 244 alleles among 88 B- and 78 R-lines, with an average of 8.7 alleles per locus, and PIC ranged from 0.17 (Xpsmp2203) to 0.91 (Xpsmp2089) with an average of 0.57; the average PIC values for B- and R-lines were 0.46 and 0.62, respectively; gene diversity varied from 0.19 (Xpsmp2204) to 0.91 (Xpsmp2089) with an average of 0.61; restorer lines (R-lines) had higher average gene diversity (0.66) than B-lines (0.50); and the R-lines had higher average heterozygosity (0.05) than B-lines (0.04). Of the 244 alleles detected in B- and R-lines, more alleles were observed in R-lines (222 alleles in 78 lines averaging 2.85 alleles per line) than the B-lines (163 alleles in 88 lines averaging 1.85 alleles per line). The neighbor-joining (NJ) tree based on a simple matching dissimilarity matrix of both the sets clearly differentiated set-I hybrid parents from set-II hybrid parents (Figure 1) into two separate clusters. 11 B-lines (11/186) were found in the R-lines cluster, while 30 R-lines (30/193) were found in the B-lines cluster. The dissimilarity matrix for 166 set-II parents clearly differentiated the majority of B- and R-lines into two separate clusters (Figure 2). Table 3. Analysis of molecular variance (AMOVA) of set-I, set-II and for combined sets using 28 SSRs. Source of variation
df
Sum of squares
Variance components
Percentage of variation
P (10,000 permutations)
Set I Between B- and R-line groups
1
256.4
1.18
16.98
0.00000
Within B- and R-line groups
424
2454.8
5.78
83.02
0.00000
Total
425
2711.2
6.97
Fixation index: 0.16981 Set II Between B- and R-line groups
1
93.2
0.53
9.22
0.00000
Within B- and R-line groups
330
1726.6
5.23
90.78
0.00000
Total
331
1819.8
5.76
1
777.8
1.58
19.20
0.00000
Fixation index: 0.092 Combined for both sets Between set-I and set-II Between B- and R-line groups within sets
2
371.8
0.95
11.55
0.00000
Among lines within groups
754
4318.9
5.72
69.24
0.00467
Total
757
5468.5
8.27
Fixation index: 0.30756
Table 4. Genetic diversity parameters of set-I and set-II lines using same set of the 28 simple sequence repeat (SSR) loci in pearl millet. SSR loci
Allele richness
Alleles per line†
PIC†
Gene diversity†
Heterozygosity†
Set-I
8.50
(1.84, 2.02)
0.59 (0.46, 0.60)
0.63 (0.50, 0.63)
0.03 (0.02, 0.04)
Set-II
8.70
(1.85, 2.85)
0.57 (0.46, 0.62)
0.61 (0.50, 0.66)
0.04 (0.04, 0.05)
†
Alleles per line, PIC, gene diversity, and heterozygosity for the B- and R-lines are given in parentheses, respectively.
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SETⅡ
0
SETⅠ
0.2
Figure 1. Unweighted neighbor-joining tree based on a simple matching dissimilarity matrix for allele sizes, detected by 28 simple sequence repeat primer pairs across 379 pearl millet hybrid parental lines (213 set-I lines and 166 set-II lines). Accessions are identified as “B” for seed parental lines and “R” as restorer lines. Suffix 1 and 2 with B- and R-lines represent set-I and set-II lines, respectively. B-lines are shown in blue and R-lines in red color.
Tree diagrams were also constructed separately for set-II B- and R-lines, which further had four sub-clusters each for B- and R-lines (Figure 3). However, 7 B-lines grouped within the R-line cluster and eighteen R-lines grouped with the B-line cluster for this set of lines. Nine of the 18 R-lines found in the B-line cluster had presence of seed parents (B-lines) in their parentage, of which 7 had the ICRISAT High Head Volume B-Composite (HHVBC) or its derived progeny (Table 5). Of the 7 B-lines found clustered with R-lines, 2 had ICTP 8202 in their parentage and one was derived from Nigerian Composite D2 Dwarf (NCD2) [26]. The 88 B-lines grouped into four sub-clusters, of which 22 lines were in cluster B-I, 15 in B-II, 24 in B-III and 27 in B-IV (Figure 3(a)). Fourteen lines having 843B in their parentage grouped in the B-I cluster. 843B is an early-maturing d2-dwarf line developed from Kansas State University (KSU) breeding line BKM 2068 [27]. Furthermore, another early promising line, ICMB 89111 derived from a cross involving 843B, was present in the parentage of 3 lines of B-I cluster. This group also had 4 lines that had progenies of Extra Early B-Composite
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R lines B lines
0
0.2
Figure 2. Unweighted neighbor-joining tree based on a simple matching dissimilarity matrix for allele sizes detected by 28 simple sequence repeat primer pairs across 88 seed parental lines (B-lines; B-1 to B-88) and 78 restorer lines (R-lines; R-89 to R-166) of pearl millet set-II.
(EEBC) in their parentage; EEBC is an extra-early-maturing and day-length insensitive maintainer composite developed using iniadi germplasm [28]. In cluster B-II, 7 lines had both 843B and 81B [29] in their parentage. Eleven lines, derived either directly from HHVBC or having its progeny in their parentage, were found very close in cluster B-III, along with 3 other such lines in this sub-cluster. Sub-cluster B-IV, with maximum number of 27 lines, had lines with mixed parentage. For instance, it had 8 lines with 843B, 13 lines with HHVBC progenies, and 5 lines with EEBC progenies in their parentages. Likewise, the 78 R-lines formed 4 sub-clusters (R-I, R-II, R-III and R-IV) comprised of 12, 8, 39, and 19 lines (Figure 3(b)). Four of the 8 lines in cluster R-II had lines derived from the ICRISAT Smut Resistant Composite (SRC) in their parentage. Of the 19 lines in cluster R-IV, 8 lines had progenies of MRC (Mandore Restorer Composite), and 5 lines had Rajasthan Composite Bajra (RCB) or Rajasthan Inbred Bajra (RIB) progenies in their parentage. Clusters R-I and R-III had lines with mixed pedigrees. ICRISAT-bred B-lines designated between 1992 and 2004 were found in the parentage of 42 of the 88 B-lines in set-II. New pearl millet composites and improved breeding populations, like the ICRISAT Medium Composite 94 (MC 94), released composite variety AIMP 92901 (based on the ICRISAT Bold-Seeded Early Composite,
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BSEC), the ICRISAT Early Smut Resistant Composite II (ESRC II), released composite variety JBV 3 (based on the ICRISAT Smut Resistant Composite II, SRC II), released composite variety Raj 171 (based on the ICRISAT Intervarietal Composite, IVC), SDMV 90031, released variety GB 8735, SDMV 95045, released synthetic variety ICMS 7704, and several RCB varieties were found in parentages of 48 of the 77 set-II R-lines.
4. Discussion 4.1. Genetic Diversity of Pearl Millet Hybrid Parents Enhancing the genetic diversity among hybrid parental lines is an essential component of hybrid breeding
BI
B II
B IV
B III
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R II
R IV R III
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Figure 3. Un-weighted neighbor-joining tree based on a simple matching dissimilarity matrix for allele sizes detected by 28 simple sequence repeat primer pairs in set-II lines. (a) Across 88 B-lines (B-1 to B-88); and (b) Across 77 R-lines (R-89 to R-166).
programs to maximize heterosis for higher grain yields. Thus, the ICRISAT-Patancheru pearl millet hybrid parent breeding program utilizes different kinds of germplasm and breeding materials in its crossing program to diversify the genetic base of the hybrid parents it develops. In the present study, the combined clustering analysis of 379 hybrid parents (213 previously designated parents of set-I and 166 newly developed parents of set-II) based on Polymerase Chain Reaction (PCR) products detected by 28 SSR primer pairs, showed 12.68 alleles per locus. This value was higher than reported earlier on pearl millet hybrid parents using SSR markers. For instance, Kapila et al. [10] detected 6.26 alleles per primer pair among 72 inbred lines (primarily B-lines bred at ICRISAT-Patancheru); and Sumathi et al. [11] reported 2.76 alleles per primer pair among 42 inbred lines bred
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Table 5. Parentage of pearl millet set-II B- and R-lines found in alternate clusters (B-lines in R-lines cluster and vice versa) for set-II lines. Acc. No.
Pedigree B-lines in R-line clusters
B-56
[(843B × ICTP 8202-161-5)-20-3-B-B-3 × B-lines bulk]-2-B-1-3-3-B-B-B-B-B
B-9
[(843B × ICTP 8202-161-5)-20-3-B-B-3 × B-lines bulk]-2-B-1
B-43
[78-7088/3/SER3 AD//B282/(3/4)EB x PBLN/S95-359]-10-2-B-2-2-B-B-B-B
B-33
[ICMB 99555 × {78-7088/3/SER3 AD//B282/(3/4)EB × PBLN/S95-359}-10-2-B-2]-22-2-1-B-B-B-1
B-20
(HHVDBC Dwarf HS-98-1-2-1-2 × ICMB 98444)-15-4-2-4
B-7
(ICMB 95111 × HTBLN/95-989035/S92-B-3)-17-1-B-B-B-B
B-24
NCD2S1-17-2-1-3-1-4-2-B-B-B R-lines in B-line clusters
R-129
Barmer × SRC II S2-112-B-2-2-2-1-6-B
R-137
ICMS 8511 S1-17-2-1-1-4-1-B-3-2-2-B-B-1
R-118
ICMS 8511 S1-17-2-1-1-4-1-B-3-2-2
R-90
GB 8735-S1-15-3-1-1-3-3-1-1-1-2-B-B
R-144
Raj 171 S1-20-4-1-1
R-126
{IP 5160-4 × (MC 94 S1-67-1-B × HHVBC)-17-2-2}-38-1-1-3
R-152
(IPC 107 × SDMV 90031-S1-84-1-1-1-1)-1-1-6-2
R-159
[((SRC II C3 S1-19-3-2 × HHVBC)-3-5-1) × (IP 19626-4-1-1-1)]-B-2-2-2-1-1-3
R-121
MRC HS-91-2-3-3-B-B-B-B-B
R-147
((ICMV-IS 94206 S1-15-2) × {(SRC II C3 S1-19-3-2 × HHVBC)-5-3-1})-B-13-4-2-1-1-1-1-1-2
R-97
HHVBC Tall S1-51-1-P1-3-B
R-163
[ICMV-IS 94206-17 × (SRC II C3 S1-1-1-2 × HHVBC)-1-3-3)]-B-9-2-3-3-1-1
R-151
(ICMV-IS 94206-7 × (SRC II C3 S1-1-1-2 × HHVBC)-1-3-3))-B-10-1-1-5-1-B-B-4
R-127
(IP 12370-1-3 × B-Lines)-B-9-1-2-1-3-3-3
R-125
[(HHVBC II D2 HS-410-1-2-4-3-1-1) × (IP 19626-4-2-1)]-B-3-1-1-1-3-3-2
R-108
[(((ICMV-IS 94206-15) × B-Lines)-B-6) × (MRC S1-156-2-1-B)]-B-22-1-3-1
R-161
[(IPC 337 × SDMV 90031-S1-84-1-1-1-1) × ICMS 8511 S1-14-3-1-1-2-B-1]-4-6-2
R-154
(IPC 1617 × SDMV 90031-S1-84-1-1-1-1)-60-2-2
primarily at Tamil Nadu Agricultural University, Coimbatore, India. The involvement of higher number of lines (379) in the present study, in comparison to the two mentioned studies (with 72 and 42 lines), might be the reason of higher average number of allele per locus in this study. However, still higher numbers of alleles per SSR primer pair (16.4) were reported in a study using 20 SSRs [30] and involving 145 pearl millet inbreds derived from diverse landrace germplasm and improved open-pollinated varieties of West and Central African origin. Similar finding of higher alleles per locus was also reported in case of maize (Zea mays L.) [31], where average of 14.57 alleles per locus was found in a study on 1537 elite inbred lines with 359 SSR markers. Twenty-eight SSR primer pairs used in the present study detected higher average number of alleles among R-lines (1.70) than among B-lines (1.27). Also, both average PIC (0.60 for B-lines and 0.73 for R-lines) and averages gene diversity (0.56 for B-lines and 0.70 for R-lines) indicated R-lines to be more genetically diverse than B-lines, which might be due to the broader genetic base of germplasm used in the development of restorer lines.
4.2. Genetic Diversity in Seed and Restorer Parents Bred over an Interval of Time Number of alleles per locus in set-II was 8.7 in comparison to 8.5 of set-I with the same set of 28 SSRs, indicating comparable genetic diversity in the hybrid parents of both the sets, and allowing us to conclude that genetic diversity within the current inbreds has not reduced compared to that in the previously designated hybrid parents.
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This established that the levels of genetic diversity are consistent in this breeding program over time. The dissimilarity matrix-based tree clearly differentiated set-I from set-II lines, indicating that new variability is being continuously infused into the ICRISAT-Patancheru’s pearl millet breeding program. New genetic stocks are regularly used in the pearl millet breeding program at ICRISAT-Patancheru to increase the genetic diversity of hybrid parents in the development of B- and R-lines. In the late 1990s and early 2000 years, there was increase in the use of already designated hybrid parents and newly developed composites in crossing program to derive new wave of genetically diverse second cycle inbreds. This was reflected from the parentages of lines under study, as new composites and improved populations, like MC 94, AIMP 92901, ESRC II, JBV 3, HHVBC, Raj 171, SDMV 90031, GB 8735, SDMV 95045, ICMS 7704, and RCB were found in the parentage of 48 of the R-lines in set-II (and none of those in set-I). Moreover, once the hybrid parents are developed, some of the promising ones amongst these first cycle parents are involved in the crossing programs to initiate another cycle of inbreeding and selection to derive new inbreds, as was reflected from set-II lines where 42 of the 88 B-lines assessed had 19 B-lines designated between 1992 to 2004 in their parentage. This approach of crossing elite inbreds is also practiced in maize to derive new inbreds [32] [33]. Contrary to our findings, reduction in the genetic diversity of new inbreds was found in comparison to the historical maize inbreds, in a study conducted on 8 new and 32 historical inbreds using 83 SSR markers [34].
4.3. Within-Line Genetic Variability in the Hybrid Parents The levels of heterozygosity detected for most of the SSR primer pairs in B- and R-lines of both the sets was within acceptable limits (
